You are on page 1of 8

Biochemical and Molecular Actions of Nutrients

Flavonoids from Almond Skins Are Bioavailable and Act Synergistically
with Vitamins C and E to Enhance Hamster and Human LDL
Resistance to Oxidation1,2
Chung-Yen Chen, Paul E. Milbury, Karen Lapsley,* and Jeffrey B. Blumberg3
Antioxidants Research Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research
Center on Aging, Tufts University and *The Almond Board of California, Modesto, CA







Almonds (Prunus amygdalus Batsch) rank highest among
tree nuts, such as hazelnut, pecan, pistachio, and walnut, in
total annual crop production (8), and are a good source of
nutrients associated with heart health such as vitamin E,
monounsaturated fatty acids, PUFA, arginine, and potassium
(9). Almond skins, removed from the nut by hot water blanching during preparation of almond meat, constitute ⬃4% of the
total almond weight and are generally treated as a waste
product. However, an array of flavonoids, including catechins,
flavonols, and flavanones in their aglycone and glycoside
forms, were identified in almond skin (10,11). These compounds may contribute to the health benefits associated with
almond consumption. The bioavailability and pharmacokinetics of individual flavonoids were demonstrated in several studies (12–14). Catechins and flavonols from food were also
found to be bioavailable in rodent and human studies with the
food, although its matrix and preparation may have a significant effect on their bioavailabilities (15–20). However, no
studies have explored the bioavailability of flavonoids from
almond skins and their effect on antioxidant activity. Therefore, we examined almond skin flavonoids (ASF)4 for their in
vitro action with and without vitamins C and E on the resistance

Flavonoids are natural constituents of plant foods and have
been carefully studied in fruits and vegetables, but less attention has been paid to their presence in whole grains and tree
nuts (1,2). Flavonoids appear to possess a variety of biological
activities, including antioxidant, anti-inflammatory, and vasodilatory actions (3). Putative health benefits of flavonoids were
suggested by epidemiologic studies showing an inverse association between intake and risk of cardiovascular disease (4,5).
A similar relation was observed with tree nuts, leading to the
recent approval by the FDA of a qualified health claim for tree
nuts, including almonds, that eating 42.6 g/d (1.5 oz/d) as part
of a diet low in saturated fat and cholesterol may reduce the
risk of heart disease (6). It is worth noting here the randomized
clinical trial conducted by Jenkins et al. (7) demonstrating
that almond consumption can reduce total and LDL cholesterol and increase the resistance of LDL to oxidation.

Presented in part at Experimental Biology 03, April 2003, San Diego, CA
[Chen, C.-Y., Shen, J., Li, T., Milbury, P. & Blumberg, J. (2003) Bioavailability
and antioxidant activity of polyphenolics from almond skins in hamsters. FASEB
J. 17: A377 (abs.)].
Supported by the USDA Agricultural Research Service under Cooperative
Agreement No. 58-1950-4-401 and the Almond Board of California. The contents
of this publication do not necessarily reflect the views or policies of the USDA nor
does mention of trade names, commercial products, or organizations imply
endorsement by the U.S. government.
To whom correspondence should be addressed.

Abbreviations used: Cmax, maximal concentration; ECD, electrochemical
detection; GAE, gallic acid equivalents, Tmax, time to maximal concentration.

0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences.
Manuscript received 13 January 2005. Initial review completed 27 February 2005. Revision accepted 23 March 2005.

Downloaded from by guest on March 10, 2014

ABSTRACT Consumption of tree nuts such as almonds has been associated with a reduced risk of coronary heart
disease. Flavonoids, found predominantly in the skin of almonds, may contribute to their putative health benefit, but
their bioactivity and bioavailability have not previously been studied. Almond skin flavonoids (ASF) were extracted
with HCl:H2O:methanol (1:19:80) and their content of catechins and flavonols identified by HPLC with electrochemical detection. ASF bioactivity was assessed in vitro by their capacity to increase the resistance of human LDL
to oxidation induced by 10 ␮mol/L Cu2⫹. ASF from 0.18 to 1.44 ␮mol gallic acid equivalent (GAE)/L increased the
lag time to LDL oxidation in a dose-dependent manner (P ⱕ 0.0001). Combining ASF with vitamin E or ascorbic
acid extended the lag time ⬎200% of the expected additive value (P ⱕ 0.05). The bioavailability and in vivo
antioxidant activity of 40 ␮mol ASF were examined in BioF1B hamsters. Peak plasma concentrations of catechin,
epicatechin, and flavonols (quercetin, kaempferol, and isorhamnetin) occurred at 60, 120, and 180 min, respectively. The concentration of isorhamnetin was significantly elevated in liver at 180 min. Absorbed ASF enhanced the
ex vivo resistance of hamster LDL collected at 60 min to oxidation by 18.0% (P ⫽ 0.028), and the in vitro addition
of 5.5 ␮mol/L vitamin E synergistically extended the lag time of the 60-min sample by 52.5% (P ⱕ 0.05). Thus, ASF
possess antioxidant capacity in vitro; they are bioavailable and act in synergy with vitamins C and E to protect LDL
against oxidation in hamsters. J. Nutr. 135: 1366 –1373, 2005.

Liver samples (0. 180.5 g cholesterol/100 g for 2 wk before the acute administration of the ASF slurry (29).59 mmol/L NaH2PO4.2 g (mean ⫾ SE). the mixture was processed in the same manner as plasma samples. Analyte separation was achieved using a Zorbax ODS C18 column (4. After overnight food deprivation. catechin. 10 – 80% (25– 60 min).5 g) was extracted twice with 10 mL acidified methanol solution (HCl:H2O:methanol. (23). deconjugation of flavonoid metabolites took longer in liver than in plasma samples.000 ⫻ g for 5 min. Due to a lower volume of ␤-glucuronidase and different buffer system. Plasma samples were collected after the whole blood was centrifuged at 1000 ⫻ g for 15 min at 4°C. pH 7. All HPLC procedures were performed with ESA instruments. BioBreeders) were used in the bioavailability experiments due to the similarity of their lipoprotein metabolism to that of humans (28). 4.4) for the in vitro LDL oxidation assay.271 ⫻ g for 90 min). Methanol was removed using a Rotavapor 134 (Buchi. approved by the Institutional Review Board of the Tufts-New England Medical by guest on March 10. After centrifugation at 14. 2014 Chemicals and reagents.4 mol/L NaH2PO4.6 mL/min flow rate and mobile phase gradient from mobile phase A (75 mmol/L citric acid and 25 mmol/L ammonium acetate in 90% H2O and 10% acetonitrile) to mobile phase B (75 mmol/L citric acid and 25 mmol/L ammonium acetate in 50% H2O and 50% acetonitrile) for 68 min. Flavonoid profile of almond skin. Whole liver was removed.01% BHT. Collection of venous blood. An aliquot of plasma was stored at ⫺80°C for determination of flavonoid status with the remainder used immediately for analysis of LDL oxidation.8 mg) was delivered in 1. All organic solvents. levels generally found in healthy people (27). sodium chloride. pharmacokinetics. were housed individually in cages with a 10:14 h light:dark cycle. (22) and expressed as ␮mol/L gallic acid equivalents (GAE). The sample was centrifuged at 1000 ⫻ g for 15 min at 4°C and methanol evaporated with nitrogen gas.0 mL saline was administered to the baseline group. The following mobile phase gradient profile was used (% solvent B): 1% (0 –15 min). and ascorbic acid was dissolved in PBS and added to the reaction immediately before initiation of oxidation. 3.79 mmol/L Na2HPO4. glacial acetic acid. and snap-frozen in liquid nitrogen for additional flavonoid analysis. and from the last 3 subjects for experiments on the interaction between ASF with vitamins C and E. METHODS AND MATERIALS to reflect amounts that can be obtained from dietary intakes (26). LDL protein was determined using a bicinchoninic acid protein assay kit (Pierce). and potassium bromide were purchased from Fisher. including 2 pumps (model 582). The supernatant was removed after centrifugation at 14. KBr and EDTA were removed from the sample using a PD-10 column (Amersham Pharmacia Biotech).0 ⫾ 1. Briefly. Spiked.5 ␮m) with a 0. The effect of ASF plus vitamin C or E on the resistance of LDL to oxidation was expressed as the lag time increase compared with the lag time of LDL without the addition of ASF or vitamin C or E.5 g) frozen in liquid nitrogen were pulverized and extracted twice with 5 mL acetonitrile containing 0. ASF with 40 ␮mol GAE (6. (24) using a Beckman NVT-90 rotor in a Beckman L8-mol/L centrifuge (329. vitamin E (␣-tocopherol). and reconstituted in 500 ␮L of 1 mol/L sodium acetate buffer. Folin Ciocalteu phenol reagent. 1. kaempferol. Concentrations ofASF from 0. sodium phosphate monobasic. The identification of individual ASF was achieved by comparing its retention time and electrochemical response with purified standards obtained from Sigma Chemical. Thus.79 mmol/L Na2HPO4. quercetin.5%. autosampler (model 542). Analysis of plasma and liver flavonoids. and 10 –1% (65– 68 min). LDL samples from the first 3 subjects were used to assess the dose-response relation of ASF.000 ⫻ g for 5 min. pH 3. Total almond skin phenolics were assessed via the Folin-Ciocalteu reaction according to Singleton et al. and reconstituted in 200 ␮L of the aqueous HPLC mobile phase. After incubation with 10 ␮L ␤-glucuronidase (98. (-)-epicatechin. The quantity of individual almond flavonoids was calculated according to concentration curves constructed with purified standards. Animals. Detection was achieved with potentials applied from 60 to 720 mV with 60-mV increments. 4. LDL was separated from plasma according to Chung et al. pH 5. and ␤-glucuronidase type H-2 (containing sulfatase from Helix pomatia). Pulverized almond skin powder provided by the Almond Board of California was used for ASF extraction. The antioxidant capacity of ASF was assessed in vitro with human LDL according to a slight modification of the method described by Esterbauer et al. ASF were extracted with 500 ␮L acetonitrile.0 mL of 0. hamsters were randomly assigned on the basis of their body weight to 6 time point groups: 0 (baseline). The powder (0. purified stan- Downloaded from jn. 2. respectively). This study was approved by the Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University. Because Ku¨hnau (30) estimated daily intake of flavonoids and other polyphenolics by a person weighing 70 kg at 14 mg/kg. Blood from each hamster was collected from the orbital sinus via micro-hematocrit tubes coated with heparin and stored in microtubes containing ETDA.0 mL via stomach gavage to hamsters anesthetized with Aerrane™ (Baxter).5. 4.6) and 20 ␮L ␤-glucuronidase (98. food-grade ascorbic acid.6 ⫻ 150 mm. Brinkmann Instrument) and the resulting slurry used for the hamster gavage. 90. isorhamnetin. was performed between 1400 and 1430 h in 6 healthy Caucasian women who were not fasting. Formation of conjugated dienes was monitored by absorbance at 234 nm at 37°C over 6 h using a Shimadzu UV1601 spectrophotometer (Japan) equipped with a 6-position automated sample changer. the women were 28 – 64 y old with a mean body weight of 63 ⫾ 15 kg. dried under purified nitrogen. The results of the LDL oxidation are expressed as lag time (defined as the intercept at the abscissa in the diene-time plot) (25).000 kU/L ␤-glucuronidase and 2400 kU/L sulfatase) were added to 200 ␮L plasma and the mixture was incubated at 37°C for 45 min. and 300 min (n ⫽ 4. rinsed with saline. Vitamin E (␣-tocopherol) was dissolved in methanol and subsequently diluted with phosphate buffer to obtain the selected concentrations. LDL (182 nmol/L) were oxidized by 10 ␮mol/L CuSO4 in a total volume of 1. combined. sodium phosphate dibasic. with a body weight of 136.0 mL phosphate buffer (7. An aliquot of ASF in acidified methanol was dried under nitrogen and redissolved in an equal volume of phosphate buffer (pH 7.59 mmol/L NaH2PO4. BioF1B strain Golden Syrian Hamsters (n ⫽ 22. and 150 mmol/L NaCl. ASF were extracted with acidic aqueous methanol as described above with the substitution of white vinegar for HCl. and Coularray 5600 A detector. Preparation of ASF for hamster gavage. 1:19:80) over 16 h at 4°C. 500 ␮L of supernatant was removed following a vigorous vortex for 30 s and centrifugation at 14. 1–10% (15–25 min). 4.4).nutrition. respectively. Antioxidant capacity of ASF in human LDL.000 kU/L ␤-glucuronidase and 2400 kU/L sulfatase) at 37°C overnight. 2. pH 7. The residue was also reconstituted with phosphate buffer (7. 100 ␮L of supernatant was injected into the HPLC.18 to 1. To increase lipoprotein production for subsequent collection. ASF and vitamin E were incubated with LDL at 37°C for 30 min before initiation of oxidation. the final concentration of methanol was 0. and 150 mmol/L NaCl. 80 –10% (60 – 65 min). we chose a test dose of 50 mg/kg body weight or 40 ␮mol GAE/hamster because rodents consume 5– 6 times more food-based energy per unit of body weight than humans (31). dried under nitrogen.4) for testing in the assay. The 1-y-old hamsters. Concentrations of vitamin E and C employed in these experiments were 9 –18% and 3– 6% of plasma concentrations. 120.ALMOND SKIN FLAVONOID BIOAVAILABILITY of human LDL to oxidation as well as their bioavailability. BHT. hamsters consumed ad libitum a nonpurified diet (Harlan) enriched with 10 g coconut oil and 0. 60. and 2. and in vivo antioxidant actions in hamsters.000 ⫻ g for 5 min.44 ␮mol/L were selected 1367 . The residue was reconstituted with the aqueous mobile phase and the ASF characterized by HPLC with electrochemical detection (ECD) according to a slightly modified method of Milbury (21). ASF in hamster plasma and liver were assessed by the HPLC-ECD as described above. 20 ␮L vitamin C-EDTA (200 mg ascorbic acid plus 1 mg EDTA in 1. All LDL experiments were performed in duplicate in each of 3 subjects. The following reagents were obtained from Sigma Chemical: copper sulfate.

3 ⫾ 3. Means without a common letter differ.05 were considered significant. Downloaded from jn. P ⱕ 0. Bars are stacked to illustrate the calculated additive effect of the 2 treatments. Because hamster LDL obtained through ultracentrifugation was lower than human LDL (determined by protein content). RESULTS The total phenolic content in the almond skins as determined by the Folin-Ciocalteu method was 8. Lag time of control (no added ASF. were added at the beginning of sample processing to construct standard curves and account for extraction losses and quantify concentrations (2).7 min. The ex vivo resistance of hamster LDL to Cu2⫹-induced oxidation was performed as described above for the in vitro experiments to test antioxidant activity of flavonoids in ASF-gavaged hamsters. The unit of the ordinate is nanoamp (nA). A paired t test was performed to determine the significance of the synergy between ASF and vitamins C or E in the in vitro human LDL oxidation by comparing the observed lag time during co-incubation with the expected (calculated) sums of values observed for ASF and vitamin C or E treatment alone. and in vitro hamster and human LDL oxidation after significant differences were obtained by one-way ANOVA.05. FIGURE 3 The synergistic effect of ASF and vitamin C or E (VC or VE) on the lag time of human LDL oxidation in vitro. Data from all time points was included in correlation tests. Differences with P ⱕ 0. vitamin E was added in vitro to hamster LDL to a final concentration of 5. 2014 dards. indicating the presence of numerous potential antioxidant compounds in almond skin (Fig.01. Based on the synergistic results obtained with ASF plus vitamin E in the in vitro experiments. 91 nmol/L LDL was oxidized by 5 ␮mol/L CuSO4 in a total volume of 1. Each trace reflects the electrochemical response of a specific applied potential (as mV) in the ECD. The difference in lag time of ex vivo hamster LDL oxidation between baseline and ASF administration was determined by a simple t test. were square root-transformed before ANOVA analysis (2). rather than an internal standard. Values are means ⫾ SE.2 ␮mol GAE/g. Lag time of control (no added ASF) ⫽ 49.5 ␮mol/L before initiation of oxidation. and kaempferol in liver. HPLC-ECD chromatography resolved ⬃30 peaks with detectable redox potential. Statistics.0001.7 min and (B) 47. 1). The recovery rates of spiked standards ranged from 75 to 80%. 1368 FIGURE 2 Effect of ASF on lag time to Cu2⫹-induced oxidation of human LDL in vitro.5 ⫾ 0.nutrition. n ⫽ 3. Labeled peaks are: (1) catechin.5 min. Five aglycone flavonoids were identified and quantified as follows FIGURE 1 HPLC-ECD profile of ASF.0 mL phosphate buffer (pH 7. n ⫽ 3. Symbols indicate different from the calculated sums: *P ⱕ 0. (3) quercetin.4). (4) kaempferol. Values are means ⫾ SE. LDL (182 ␮mol/L) was oxidized by 10 ␮mol/L Cu2⫹ with the addition of ASF. The percentage value above the solid bar indicates the observed synergy greater than the calculated sums of the individual treatments. The JMP IN 4 statistical software package (SAS Institute) was used to perform all statistical analyses. The TukeyKramer honestly significant difference test was used in experiments on ASF in hamster plasma. All results are reported as means ⫾ SE. including epicatechin and isorhamnetin in plasma and catechin. (2) epicatechin. or VE) ⫽ (A) 45. quercetin. VC or VE. or ASF ⫹ VC or VE. Ex vivo antioxidant capacity of ASF.CHEN ET by guest on March 10. (5) isorhamnetin.005.7 ⫾ 1. †P ⱕ 0. ‡P ⱕ 0. . The equal variance assumption was assessed by Hartley’s test (32) and data. VC. epicatechin.

01). and kaempferol (3.nutrition. respectively. In vitro. respectively. (4) kaempferol. 3A). ASF increased the resistance of human LDL against Cu2⫹-induced oxidation in a dose-dependent manner (P ⱕ 0. Values are means ⫾ SE. quercetin (6. epicatechin. and 761 nmol/L. 222.9 ⫾ 1. (2) epicatechin. respectively.. (nmol/g): catechin (186.75 and 5. (5) isorhamnetin. 4).5 ␮mol/L vitamin E (P ⱕ 0. All 5 identified flavonoids from almond skins were bioavailable in hamster plasma (Fig.9). 2014 FIGURE 4 HPLC-ECD chromatographs of hamster plasma samples obtained at 60 min (A) and 180 min (B) after administration of 40 ␮mol GAE ASF and immediately after gavage with saline (baseline). isorhamnetin (20.3 ⫾ 0.5 ␮mol/L increased lag time by 19. and isorhamnetin in the plasma of hamsters administered 40 ␮mol GAE ASF. 2). epicatechin.36 ␮mol/L of ASF in combination with 5.5). . quercetin. Based on their pharmacokinetic profile in the plasma. Ascorbic acid at 2.1 ⫾ 1.6 and 34 ⫾ 2. The time to reach the Cmax (Tmax) was 60 and 120 min for catechin and epicatechin. the maximum concentrations (Cmax) of catechin. kaempferol.18 and 0. an observed value twice the calculated additive value) was observed with 0. 72. epicatechin (77. no synergy was observed with the 2. vitamin E at 2. n ⫽ 4.7 min. The lag time was 289% longer than the expected additive value with the combination of 0.0001) (Fig. and 180 Downloaded from jn.36 ␮mol/L ASF and 5.6% of the total phenolic content of almond skins. (A) 210 mV ECD trace.e.0 ␮mol/L increased lag time by 12. These 5 flavonoids represent 3.9). and FIGURE 5 Time course of catechin.0). 5).5 and 5. quercetin.001) (Fig.5 ␮mol/L dose of vitamin C (Fig. P ⱕ 0.7 min.ALMOND SKIN FLAVONOID BIOAVAILABILITY 1369 isorhamnetin were 376. respectively ( by guest on March 10. kaempferol. A 1-fold synergy (i.0 ␮mol/L ascorbic acid (P ⱕ 0. 133. 3B).4 and 24. (3) quercetin.8). Labeled peaks are: (1) catechin. (B) 70 mV ECD trace.05. Means in each panel without a common letter differ.

5. 3). respectively). kaempferol.31 — — ⱕ0.0001 — — — 0. 182.54 ⱕ0. If a prooxidant action of quercetin occurred in our study. in contrast to the 180 min observed in plasma. and flavonols (10.0. 120. The bioavailabilities of selected single catechin and flavonol compounds were reported (14. DISCUSSION The putative health benefits of flavonoids have been attributed in part to their antioxidant activity (33.0001 — — 1 Based on one observation in 22 hamsters.7 min.9 ⫾ 2. The Cmax of isorhamnetin in the liver was 698 nmol/g at 180 min after administration. Absorbed ASF appeared to induce a small increase of 18.3 ⫾ 17. 1).org by guest on March 10.11).2 0. 2) (26). flavanones. Almond skins were previously reported to contain 3 classes of flavonoids.66 0. 38.16..8 ⫾ 0. (40) suggested that polyphenolics may stabilize the LDL particle structure via an interaction with the apoprotein-B domain.3 ⫾ 1. (38) reported a prooxidant activity of low concentrations of quercetin (⬍2 ␮mol/L) indicated by increased malondialdehyde formation during Cu2⫹-induced LDL oxidation. isorhamnetin.66 to 0. it may have been masked by other constituents of almond skin. Similar to our finding in hamsters.0. 160. and p-hydroxybenzoic acid (10. and quercetin and urate (42).5% longer lag time than that collected at baseline (P ⱕ 0. However.8.40. vitamin C may contribute to the synergy by inhibiting the decomposition of lipid peroxides and/or preventing Cu2⫹ from binding to LDL (40). quercetin. However.44). Further. 2014 min for the 3 flavonols. The lag time of ex vivo hamster Cu2⫹-induced LDL oxidation was 30. except for isorhamnetin. we examined the behavior of a complex array of polyphenolics in almond skins while measuring several specific flavonoid constituents.4. and kaempferol in liver were not affected by acute ASF gavage.0001 ⱕ0. Such interactions (14. Hwang et al. others found a faster plasma Tmax for catechin than quercetin when single compounds were fed to rats (16. only LDL collected at 60 min showed a synergistic increase with a 52. such as glycosides of quercetin.73 — ⱕ0.CHEN ET AL. 1370 TABLE 1 Correlation coefficients among flavonoids in the plasma and liver of hamsters administered 40 ␮mol GAE ASF1 Kaempferol Plasma Quercetin Kaempferol Catechin Liver Quercetin Kaempferol Catechin Plasma and liver Isorhamnetin Isorhamnetin Epicatechin r P-value r P-value r P-value 0. In contrast. the plasma concentrations of these flavonoids were less than half the Cmax and their concentrations (with the exception of quercetin and isorhamnetin) did not differ from the baseline value. These results are consistent with several reports of the antioxidant activity of flavonoids such as catechin and quercetin with concentrations administered ranging from 0. The epicatechin concentration was significantly elevated at 300 min.0 and 24. the lag time of LDL oxidation was 119.2 ⱕ0. kaempferol. but not quercetin.1 ⫾ 1. differed from those in the plasma (Fig. epicatechin.05).01 — — 0. . likely flavonoids or related polyphenolics. The Cmax of liver quercetin and kaempferol at 73 and 130 nmol/g did not differ from baseline and their Tmax was 90 min. When 5. and 38.78 although no relation was noted between the catechins (Table 1).9 min at the baseline.34 — 0. Isorhamnetin showed a similar pharmacokinetic pattern in both plasma and liver.1 ⫾ 8. catechins.8. 6).0% in the ex vivo resistance of LDL to oxidation at 60 and 120 min (P ⫽ 0. vanillic acid.5 ⫾ 1. and 160. Using HPLC-ECD. 34. i.0001 — — 0. and isorhamnetin) and measured ⬃25 other redox compounds (Fig.05). suggesting that catechin is rapidly absorbed Downloaded from jn.19.11). At 300 min.0 ⫾ 6.028 and 0. Correlation coefficients between the 3 plasma flavonols ranged from 0.nutrition. It is possible that the potential prooxidant actions of single flavonoids do not occur in natural mixtures of plant polyphenols in which opportunities for recycling oxidized compounds may exist. 90. we quantified 5 aglycones (catechin. such as the regeneration of oxidized malvidin 3-glucoside by catechin (39). Pharmacokinetic patterns of the flavonoids in liver.78 — — ⱕ0. 60. and naringenin as well as protocatechuic acid. 36.34). quercetin.4.08 0.9 ⫾ 3.0001 — — 0. isorhamnetin remained higher than baseline levels after 300 min (P ⱕ 0. and 180 min time points. In contrast.25 to 10 ␮mol/L (35–37). Isorhamnetin pharmacokinetics paralleled those of kaempferol.62 — — ⱕ0.43.9 ⫾ 13.7.19 — — 0.008. most of the supportive evidence has been based only on in vitro experiments or in vivo feeding studies with a single flavonoid aglycone or glycoside (26). Concentrations of quercetin and kaempferol were correlated (Table 1). oat phenolics and ascorbic acid (2).44).5 ␮mol/L vitamin E was added in vitro to the reaction. respectively. Concentrations of catechin. Extrapolations about flavonoids from in vitro results are limited because of their relatively poor general bioavailability and extensive biotransformation in vivo (14). Filipe et al.e. but little information is available regarding the concurrent absorption profile of mixtures. 152.005 — — — 0. respectively. The complex of ASF effectively increased the resistance of human LDL to oxidation in vitro in a dose-dependent fashion within physiologically relevant concentrations (Fig. In addition to recycling mechanisms. Similar synergies were observed between genistein and ascorbic acid (40).41) may also partly account for the synergy between the ASF and vitamins C and E observed here (Fig.

quercetin. in humans (26). The plasma pharmacokinetics of the almond flavonols were similar with high correlation coefficients (Table 1). it was not associated with other hepatic flavonols. especially epicatechin. The increase in hepatic catechins. although the vitamin E content of the nut may have contributed substantially to this result. The relatively high level of isorhamnetin in liver may reflect a slow rate of clearance compared with other flavonoids or a gradual contribution from methylation of aglycone and glycone quercetin. a very modest antioxidant effect of ASF was suggested by the small increase in the resistance of LDL in hamsters treated with the almond skin extract compared with saline. In contrast to a Tmax of 120 min for epicatechin in hamsters (Fig. we found a shorter half-life in plasma for the catechins compared with the flavonols in hamsters. although such differences could be accounted for by differences in their glycosides (12). 5). Consistent with the antioxidant capacity of almond skins using the LDL oxidation assay. Means in each panel without a common letter differ. (14) reported that ⬎90% of catechin and quercetin in rat liver was methylated. No association was noted between the plasma concentrations of the 2 catechins. n ⫽ 4. respectively. at 300 min may reflect an unusually slow distribution to this tissue or result from the small sample size employed at this one time point. (46) demonstrated the antioxidant potency of whole almonds in vitro with the ferric reducing antioxidant power assay. such as quercetin. 2014 FIGURE 6 Catechin. kaempferol. and isorhamnetin in the liver of hamsters administered 40 ␮mol GAE ASF. Halvorsen et al. with the exception of isorhamnetin. epicatechin. Although the number of reports is limited. e.05. epicatechin. and kaempferol in hamster liver may be attributed to their presence in the basal nonpurified diet fed to hamsters. Although isorhamnetin is a metabolite of quercetin (45). caution is warranted when interpreting these values due to pharmacokinetic differences between species as well to other confounding variables such as differing ingredients in the diet fed and potential competition between polyphenolics for absorption (44). Although a higher dose may have produced a greater effect. Manach et by guest on March 10.g. Similar to the relations suggested by Manach et al.. suggesting the potential contribution to this value from unidentified flavonol glycosides in almond skins. 5). quercetin. P ⱕ 0.5 and 7 h. kaempferol. The half-life of flavonols in the hamsters (5 h) was shorter than that in rats (11–28 h). 1371 . or quercetin in the liver. the weak direct response elicited in vivo may be a result of a lower potency of glucuronidated. sulfated. quercetin-4⬘-glycoside and rutin (quercetin rutinoside) have Tmax of 0. quercetin and kaempferol concentrations in the liver were correlated. although it is possible that these flavonoids were quickly redistributed to other tissues. However. the association between their plasma concentrations was low. (44) reported a 60-min value in rats. Similar to their relation in plasma (Table 1). Although isorhamnetin status in plasma was correlated with its concentration in liver. the 2 catechins and 3 flavonols were detected in liver although.11). Noticeable amounts of catechin. and kaempferol glycosides (10. glycoside moieties appear to have a substantial influence on pharmacokinetics. Baba et al. and/or methylated metabolites of the ASF. possibly due to direct contribution of isorhamnetin from almond skins. It is not clear why ASF administration did not increase catechin. Values are means ⫾ SE. Indeed. In vivo.ALMOND SKIN FLAVONOID BIOAVAILABILITY in the upper gastrointestinal tract and cleared from plasma within several hours. (13) for humans. 6). In addition. We observed a Tmax at 180 min for the 3 almond skin flavonol aglycones after deconjugation by glucuronidase and sulfatase in hamsters (Fig. Moon et Downloaded from jn. In hamsters. isorhamnetin.nutrition. after ASF gavage. their pharmacokinetic patterns were quite different from those in plasma (Fig. flavonoids were detected in mouse and rat tissues with concentrations from 30 to 3000 ng aglycone equivalents/g (13).

A. 50: 2459 –2463. J. Chung. R. Q. Frison-Norrie. 6.. Maziere. Mengelers. Biochem. Respir. Nutr. L. Demigne´. Downloaded from jn. It may be attributed to the more potent antioxidant activity of catechin against copper-induced LDL oxidation than quercetin (49). Osgood. C. 16.. & Jimenez. Clin. & Rosen. J. Hider. 9.. M. S.. 9. Sang. K. J. J. Marchie. Med. Milbury. W. & Sporns. Clin Nutr..nutrition. Rissanen. Ku¨hnau. 2005]. L... Ho. A class of semi-essential food components: their role in human nutrition. N. J.usda. Agric... Y. Med. 19. and lovastatin on oxidative status and aortic fatty lesions in hyperlipidemic-diabetic hamsters. Soc.S. Of the 5 identified flavonoids in the ASF. Nutr. K. C. (2002) Dose response of almonds on coronary heart disease risk factors: blood lipids. J. G. C.W. 27. lettuce and celery. Collins. (2004) Bioavailability of quercetin in pigs is influenced by the dietary fat content. P. 13: 341–390. B. Y. Department of Agriculture. 37. S. Because almonds represent one of the richest dietary sources of vitamin E. Drake. Biochem. Commun.. 13. Ott. B. Soc. onions.. (1993) An Introduction to Statistical Methods and Data Analysis. Nuts. McDonald. by guest on March 10. A. 32. Connelly.. Zhu. Helio¨vaara.. R.. Clin.. Fernandes. L. Interestingly. rather than at 180 min. (2000) The effect of vitamin E. A. Food and Drug Administration (2003) FDA Authorizes Heart Disease Health Claim for Nuts. Am. Maziere. J... 33. 21: 284 –291. Biochem. 22. 35. J..nal. Phytochemistry 65: 2391–2399. C.A. M. (1995) Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Wadsworth Publishing Co. (1996) Role of dietary flavonoids in protection against cancer and coronary heart disease. Regerat. P.. Nutr. Proc. D. van Norren. J. C.. the ASF-vitamin E synergy appeared in LDL collected at 60 min. (2002) Antioxidative phenolic compounds isolated from almond skins (Prunus amygdalus Batsch).. almonds [Online]. Ma.. vd Gaag. J.. W. C. Quercetin as an effective antioxidant against pro-oxidant effects of urate. R. G. E.. van het Hof. 14. Mengelers. M. J. Nutr.. E. J. Schunemann. & Oka. H. Lachance.. Huxley.. P.ers. Smith. J. Free Radic. Silva. Milbury. Texier. Clin. & Chen. ASF possess antioxidant capacity and interact with vitamins E and C in a synergistic manner to protect LDL against oxidation in vitro. M. Agric. Clin.. Sanna. L. retinol and carotenoids with pulmonary function in the general population. C. despite a weak effect of ASF on the resistance of LDL to oxidation ex vivo. 38. R. & Jurgens. Cermak. Z. Economic Research Service of US Department of Agriculture. T. M. Filipe. USDA National Nutrient Database for Standard Reference. J. & Wolffram. controlled. S. & Rice-Evans. Nonetheless. T. J.. van Leeuwen. Qian. Food Chem. FTS2004 [Online]. M. & Re´me´sy. 1372 ACKNOWLEDGMENTS We thank Donald Smith for his contributions to establishing the hamster model and Ting Li and Jennifer O’Leary for their excellent technical assistance in the laboratory.. (2004) Polyphenolics: food sources and bioavailability. Eur. Lou. 2005]. P. Nutr. J. M. Nutr. P. S. J. Manach. P. H. E.. 220: 198 –202. C. J. Reuanen. G.. G. K. & Katan... 24: 785–789.. the Tmax for catechin. Jenkins. L... Hakulinen.. Vidgen. D. Biol. A.. H. 45: 590 –595. Med.20). S. Med. Am. homocysteine. Yang. P. (2002) Red wine and black tea polyphenolics modulate the expression of cycloxygenase-2. Semanm. U. B. (2005) Bioavailability and bioefficacy of polyphenols in humans. Docket No. Free Radic. 02P-0505. (1999) Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin.. P.. H. & Katan. T. World Rev. Biol. C. Wiseman. G. C. 47: 2020 –2025. A.. R.usda. C. Favier. Manach. J. (1997) Quantitative analysis of the flavonoid content of commercial tomatoes. (1995) Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. S. G. F. 30. Faulkner. Manach. J. (1980) Preparative and quantitative isolation of plasma lipoproteins: rapid. Atherosclerosis 149: 277–286. J. Yuan. Nutr. LITERATURE CITED 1. 4. El-Sweidy. G. B. 81: 230S–242S. H. 11. Parker. & Blumberg. Singleton. 34. V. Ren. Esterbauer. 2. H. (2001) Flavonoids: a review of probable mechanisms of action and potential applications. Clin. its antioxidant action was strengthened by their synergy with the vitamin E added in vitro in this experiment. Biol.. (2004) Anti. when the greatest concentration of flavonols was obtained. C. FEBS Lett. further research is warranted examining the relations between the natural ingredients in this whole food and their potential association with health benefits. 134: 1508 –1511. and their antioxidant capacity was amplified with the in vitro addition of vitamin E. Huang. Texier. H. J.. C. O. E.. H. Methods Enzymol. 28.. Williamson. D. & Neil. they may be less available to interact with LDL (16. P. 20. Trans. Diet. Boelens. R.. Y. Crespy. 62: 1276 –1282. & Segrest. M. F. V.. 21: 703–707. (1992) The role of lipid peroxidation and antioxidants in oxidative modification of LDL. C. J. M.. S. 50: 2782–2787. Texier.. 26. 15. Grant. Food Chem. & Meydani.. van Nood.. C.. Browne. 9.. Lesser..html [accessed Mar. 76: 560 –568. Crit. Y. (2003) The relation between dietary flavonol intake and coronary heart disease mortality: a meta-analysis of prospective cohort studies.. & Katan. 8. (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.. (1997) Bioavailability of rutin and quercetin in rats. J. W. Klocke. R. Luceri. Biol. Santus. J. Jeong. F. M. Demigne´. Hertog. Regerat. Morand. J. H. lipoprotein(a). J. Needs. S. R. C. 134: 1459 –1466. . 38: 877– 884. L. de Vries. Tsang.. Eur. Samuel. Am.. A similar synergistic relation was observed between oat phenolics and vitamin C in the same hamster model (2). E. Freudenheim. Lapsley. 335: 15–26. (1999) Tea flavonols in cardiovascular disease and cancer epidemiology. Biol.S. P. J. Food Chem. S. Caderni. Review of 97 bioavailability studies. Morand.. 271: 1991–1999. Agric. S. J. http://www. & Rotheneder. M. B. Agullo. Free Radic. & Black. van Hoorn.. Regerat. (1976) The flavonoids. L. A.. B. J. Proc.. (2004) Avenanthramides and phenolic acids from oats are bioavailable and act synergistically with vitamin C to enhance hamster and human LDL resistance to oxidation. M.. J. C. such as a reduction in the risk of cardiovascular disease. Hollman. P. C. D. Demigne´. 3. 29. van Dongen. El-Swefy. J.. & Trevisan. Khodr. & Lamuela-Raventos. inducible nitric oxide synthase and glutathione-related enzymes in azoxymethane-induced F344 rat colon tumors. (2004) Polyphenols from peanut skins and their free radical-scavenging effects. J. A. Free Radic. W. C.. B. Freitas. (1999) Plasma and lipoprotein levels of tea catechins following repeated tea consumption. van Trijp. 24. Gebicki. C. A. R.. M. (1996) Absorption and disposition kinetics of the dietary antioxidant quercetin in man. Fruit and tree nuts outlook. J. (2002) Flavonoid intake and risk of chronic diseases. O. E. P. M. S. Release 17 (2004) Agricultural Research Service.... P. 133: 4183– 4188. Sevanian. Janisch. Muti. 5. C. G. [accessed Mar. Exp. 299: 152–178. 25. D.. O. J.. C.. & van Leeuwen. Puhl. Soc. 220: 203–209. P. J. C. W. oxidized low-density lipoproteins. M. (2001) Analysis of complex mixtures of flavonoids and polyphenols by high performance liquid chromatography electrochemical detection methods. Geer. 36. J. Belmont.. de Vries. G. Exp. Crozier. K. & Re´me´sy. Re´me´sy. 17. & Morliere. R. Methods Enzymol. 7. probucol. T. D. 132: 1376 –1379. Smith. E. & Re´me´sy. et al. and pulmonary nitric oxide: a randomized. C. Manach. Agric.. Further. Ordovas. Circulation 106: 1327–1332. all appeared in plasma and liver after oral administration. Schaefer. Morand. Williamson... 18. A. (2001) The relation of serum levels of antioxidant vitamins C and E. J. & Katan. Haight. Food.. single discontinuous density gradient ultracentrifugation in a vertical rotor. Am. (1989) Continuous monitoring of in vitro oxidation of human low-density lipoprotein. P. H. (2004) Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin. W.J. 2014 al. & Re´me´sy. Orthofer. J. Ja¨rvinen. Res. Haigle. Brown.E. 27: 1259 –1266. I.. M. U. C. C. Am. K. Wilkinson. B.. H.. (1999) Comparison of the bioavailability of quercetin and catechin in rats.. & Aromaa. M. Nijveldt. H.. J.. R. C. Nutr.. C. Knekt. P. 31. M. E. Free Radic. Scalbert. A. Res.. Hollman. (1998) Structural dependence of flavonoid interactions with Cu2⫹ ions: implications for their antioxidant properties. Med. Feskens.. A. In summary.. P. C.CHEN ET AL.. & Tijburg. P. & Plumb. Esterbauer. A. (2002) Identification and quantification of flavonol glycosides in almond seedcoats using MALDI-TOF MS. M. H. R. A. 24: 117–191. D. Dorfman.... 74: 418 – 425. 330: 1173–1178. 23. B. R. H. J.E. C. F. J. Morand.. Kwak. M. Care Med. S. (2003) Study of diet-induced changes in lipoprotein metabolism in two strains of GoldenSyrian hamsters. Lean. 10.and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation. Scalbert. & Lichtenstein. Nutr. 57: 904 –908. (47) and Cren-Olive et al. H. These flavonoids and/or related compounds in the ASF slightly enhanced the resistance of hamster LDL against ex vivo Cu2⫹-induced oxidation. CA. 79: 727–747. J. 409: 12–16... J.. E. Manach. Nutr. Chem. Kumpulainen. M.. Lipid Res. L.. A. crossover trial. 6: 67–75. Striegl. C. Chen. P. D. Puhl. D. 12. C. J. 21. Nutr.. many flavonoid metabolites appear bound to plasma proteins in vivo.C. E. P. D. Am. & Dolara. Morand. http://www.. (48) both reported that conjugated derivatives of quercetin and catechin provided less antioxidant protection than their parent compounds against LDL oxidation in vitro. Kendall. Hollman. W. 125: 1911–1922. Ji. H.. A. S. T. C. thus. 163: 1246 – 1255..

V.. Am. Biochem. K.. Holte. 323: 373–381. Besson. J. Vieira. & Almeida. 43. A. Med. Kido. Baba.. J. M. Biochem.. Nakahara.nutrition. Mattivi.. A. E.. Madeira. Hwang. Volkova. Haffner. Duriez. a water soluble flavonoid. Cell.W.. & Ursini.. Shaalan. Usui. Nutr. 131: 2885–2891. Med.. Biophys. Morand. Manach. Arch. Tsushida. C. 29: 79 – 89. Teissier. Y. A.. M. & Re´me´sy. J. T.. & Terao.. I. Biophys. M. Y..C. Biol.. F.. 40. Remberg. (2003) Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy. Morliere. J. C.. V. C. N. Goldblum. Lunelli. (2001) Flavonoids and urate antioxidant interplay in plasma oxidative stress. & Fernandes... 41. J.. and alphaG-rutin. Baugerod. Demigne´. & Rolando.. & Rigo.. P. Biol. Mahmood. 48. 47. Yoshizumi.. Free Radic. S. (2003) Absorption and urinary excretion of quercetin. 42. K.. Free Radic. Hodis. 408: 239 –245. 2014 ... in rats. Scarpa. Biol. (2002) Synergistic antioxidant effect of catechin and malvidin 3-glucoside on free radical-initiated peroxidation of linoleic acid in micelles.. by guest on March 10. T.. (2000) Synergistic inhibition of LDL oxidation by phytoestrogens and ascorbic acid. (2001) Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin. Myhrstad. N. J. C. Musa. L. Arch. H. M. Natsume. H. L. 44. Agric. Vaya. Wold. Mol. 34: 850 – 855. S. N.ALMOND SKIN FLAVONOID BIOAVAILABILITY 39. S. A.. V.. & Terao.. 49. 275: R212–R219. A. 221: 79 – 87. Vanzani. 1373 (2001) In vivo comparison of the bioavailability of (⫹)-catechin. Takizawa. Physiol. Moon. T. Laranjinha. Aviram.-B. (1995) Two related phenolic antioxidants with opposite effects on vitamin E content in low density lipoproteins oxidized by ferrylmyoglobin: consumption vs regeneration. J. C. Cren-Olive. F. J.. Muto. K. B.. rutin. 45. Shimoi. P. F. Crespy. Hvattum.. Downloaded from jn. A. N.. J... & Yumoto. C. Santus. Lanca. O. R. Filipe.. et al.. S. K.. Osakabe. & Tamir. T. P. Phytochemistry 62: 89 –99. Food Chem. E. Med... Biochem. (-)-epicatechin and their mixture in orally administered rats. M. (1998) Plasma metabolites of quercetin and their antioxidant properties. Nutr. 51: 2785–2789. C. (2002) A systematic screening of total antioxidants in dietary plants. Silva. P. Free Radic. H. Barikmo. 46. Sevanian. J. Rossetto. M. 30: 1274 – 1285. 132: 461– 471. J. K.. R. (2003) Effect of catechin O-methylated metabolites and analogues on human LDL oxidation.